Cloning of CgYPS7gene

stranded DNA. Final reaction volume was adjusted to 20 μl with DEPC-treated waterandamplificationreaction was carried out usingthese parameters: initial denaturation at 95 ̊C for 5 min followed by 40 cycles of denaturationat 95 ̊C for 30 sec, annealing at 55 ̊C-57 ̊C for 30 sec, elongation at 72 ̊C for 40 sec and final extension at 72 ̊C for 10 min. Transcript levelswerequantified with an end-point value known as Ct (cycle threshold). The Ctdefines the number of PCR cycles required forthe fluorescent signal of SYBR green dye to cross more than the background level. The Ctvalue isinversely proportional to the amount of nucleic acid product. Ctvalues were obtained during exponential phase of amplification and used forcalculation of relative-fold change in gene expression after normalization to Ctvalues ofeither housekeeping gene ACT1 (gene encoding actin)orTDH3 (gene encoding Gapdh)with the help of the following formula. Fold change in expression = 2-∆∆Ct∆∆Ct= ∆Cttreated -∆Ctuntreated∆Cttreated = Ctvalue forgene of interest under test/treatedcondition -Ctvalue forinternal controlgene(ACT1/TDH3) under test/treatedcondition∆Ctuntreated = Ctvalue forgene of interest under untreatedcondition -Ctvalue forinternal control (ACT1/TDH3)gene under untreatedcondition

Todeterminethe expression level of a specific gene, quantitative real-time polymerase chain reaction (qRT-PCR/qPCR)was performed oncDNA usinggene specific primers. Primers for qPCR weredesigned in such a way so as to get amplification products in a size range of 150 to 300 bp. Optimalprimer and cDNA concentrationswere standardized and qPCR was performed in ABI Prism 7000/7500 Real time PCR Machine (Applied Biosystems). Briefly, 0.4 μl cDNA was mixed with 0.1 to 0.2 picomolesof gene specific forward and reverse primers and 10 μl 2X MESA GREEN qPCR™Mastermix Plus containing SYBR green dye (Eurogentec) in awell of a96-well PCR plate (Axygen). SYBR green is a dye that specifically binds to double

Quantitative real-time polymerase chain reaction (qRT-PCR)

1 μg good quality RNA was treated with DNase I (amplification grade, Invitrogen) to remove DNA contamination and used for complementary DNA (cDNA) synthesis using reverse transcriptase enzyme and oligo-dT primers.SuperScript®III First-Strand Synthesis System (Invitrogen) was used to carry out cDNA synthesis reaction according to the manufacturer’s instructions. cDNA was stored at -20 ̊C

Complementary DNA (cDNA) synthesis

autoclavable plastic items to removeRNase contamination. RNA was isolated from C. glabratacells using hot phenol extraction strategy.Log-phase cells well harvested at 5,000 g for 5 min at 4 ̊C, resuspended in 1 ml ice-cold DEPCwater and transferred to a 2 ml microcentrifuge tube. Cells were spun down at 6,000 g for 3 min at 4 ̊C and resuspended in 350μl AEsolution. 50 μlSDS and 400 μl acid phenol wereadded tothe above tubeand mixed well by vortexing. Tubes were incubated at 65 ̊C for 15minwith continuousmixing. After incubation, tubes were kept on ice for 5 min and centrifuged at 12,000 rpm for 5 min at 4 ̊C. Aqueous phase was collected and re-extracted with an equal volume of cholroform. Total RNA was precipitated at -20 ̊C with1/10thvolume of 3 M sodium acetate (pH 5.2) and 2.5 volume of ice-cold 100% ethanol and collected by centrifugation at 12,000 rpm for 5 min at 4 ̊C. RNA pellet was washed with ice-cold 70% ethanol and resuspendedin 100 μl commercially available DEPC-treated water (Sigma # 95284). RNA concentration was measured byrecordingabsorbance at 260 nm. Purity of RNA sample was checked by A260nm/A280nmratio where ratio of >1.8 was considered as good quality RNA. RNA integrity was checked by gel electrophoresis on 8% agarose gel made in DEPC-treated TAE buffer

All solutions were made in RNase free diethylpyrocarbonate(DEPC)water. Microcentrifuge tubes and tips employed for RNA workwere autoclaved twice and kept at 70 ̊C for overnight before use. RNaseZap®(Ambion) was sprayed on non-

Total RNA isolation

Themethod was used for isolation of good quality genomic DNA that wasused to map Tn7insertionin C. glabratamutants.Briefly,10 mlsaturated yeast culturewasharvested, resuspendedin 1 ml sterile water and transferred toa2 ml microcentrifuge tube. Cells were pelleteddown by centrifugation at 4,000 rpm for 5 min. Supernatant was discarded and the pellet was resuspendedin 500 μl freshly prepared solutioncontaining100mM EDTAand 5% β-mercaptoethanol andincubated at 42 ̊C for 10 min. After incubation,cells were spun down at 5,000 rpm for 1 minand resuspendedin 500μl freshly-prepared BufferB. One tip full of lyticase(Sigma # L4025) was added and cellsuspension was incubated at 37 ̊C for 1 h. Following incubation,cell suspension was spun down at 6,000 rpm to recover spheroplasts.Spheroplasts weregently resuspendedin 500μl BufferCand DNA was twice extracted with 500μl phenol:chloroform:isoamyl alcohol (25:24:1)solution.Aqueous layer was collected in a new 2ml microcentrifuge tube and DNA was precipitated with 1ml ethanol and 1/10thvolume of 3M sodium acetate (pH 5.2)by centrifugation at 13,000 rpm for 5 min. Pellet was resuspendedin 200 μl TE containing 0.3 μl of RNase Cocktail™and incubated at 37 ̊C for 30 min.After incubation, 300 μl additional TE was added and DNAwas re-precipitated withethanol and 3 M sodium acetateas described above. Pellet was washed with 70% ethanol anddried under air. DNA pellet was finally suspended in 100 μl TE and stored at -20 ̊C

Protocol III(Spheroplast lysis method

phenol:chloroform:isoamyl alcohol (25:24:1)was added to the tube and mixed thoroughly.Aqueous phase was collected after centrifugationat 12,000 rpm for 3 minand was transferred toanew 2 ml microcentrifuge tube.1 ml absoluteethanol was added to the aqueous phase and DNA was precipitated by centrifugation at 12,000 rpm for 8 minat 4 ̊C.DNA pellet was washed with chilled 70%ethanol and dried under air. DNA pellet was resuspendedin 50 μl TE containing 0.3 μl of RNase Cocktail™(Ambion®# AM2286)and incubated at 50 ̊C for 20 min. 200 μl additional TE was added to the above suspension and DNA was stored at -20 ̊C

In this method of genomic DNA extraction,yeast cells werelysed by mechanical disruption with glass beads. Briefly, yeast cells were harvested after overnight growth in YPD medium, resuspendedin 500 μl waterand transferred toa2 ml microcentrifuge tube.Cells were pelleteddown at 10,000rpm for 1 min. Resulting supernatant was discarded and the pellet was resuspendedin 500 μl Buffer A. The tube was incubated at 65 ̊C for 15 min. After incubation, 500 μl ofphenol:chloroform:isoamyl alcohol (25:24:1) and 0.5 gm of acid-washed glass beads (Sigma # G8772) were addedto the tube. Cells were lysed by three cycles of high speed vortexing withintermittent ice breaksfor 45 secand pelleteddown at 12,000 rpm for 3 minat 4 ̊C.Uppermost aqueous phase was transferred to a 2 ml microcentrifuge tube,500 μl of

Protocol II (Glass bead lysis method)

This quick extraction method was used to isolate genomic DNA which was used as templateto amplify gene of interestor toverify the knock-out. C. glabratacells were grownovernight to saturation in 10 mlYPD medium at 30 ̊C.Cells were harvested at 4,000 rpm for 5 min, resuspendedin 400 μl Buffer Acontaining 50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1%Triton X-100 and 1%SDSand weretransferred to a2 ml microcentrifuge tube. Equal volume ofphenol-chloroform solution was added to the abovesuspensionfollowed byvortexingfor 2-3 minand incubationat 42 ̊C for 30 minwithcontinuous agitation at 800 rpm on thermomixer (Eppendorf). Cell debris was removed bycentrifugation at 12,000 rpm for 5 minand aqueous fraction(~ 350 μl)was transferred to a new 2 ml microcentrifuge tube.0.3 μl RNaseCocktail™(Ambion® # AM2286) containing RNase A (500 U⁄ml) and RNase T1 (20,000 U⁄ml) was added and tubes were incubated at 37 ̊C for 30 min. DNA was precipitated with 2.5 volumesof chilled ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNA pellet was washed with chilled 70%ethanol and semi-dried under air.Pellet was suspendedin 100μlTE (10 mM Tris-HCland 1 mM EDTA; pH 8.0)and stored at -20 ̊C.DNA concentration was determined by recordingabsorbance at 280 nmin Nanodrop (Nanodrop ND-1000, Thermo Scientific).

Protocol

Based on the subsequent use, DNA from C. glabratacells was extracted using three different methodologie

Yeast genomic DNA isolation

Molecular biology tech

C. glabratayeast cells were grown overnight in 5 ml YPD medium at 30 ̊C. An aliquot from the overnight culture was inoculated in 10 ml fresh YPD medium to an initial OD of 0.1. Cells were incubated at 30 ̊C till the cultureOD600was between 0.4 and 0.6. Cells were harvested in a sterile 50 ml centrifuge tube and washed twice with sterile Milli-Q(MQ)water. Washed cells were suspended in 100 μl of 100 mM LiOAc, mixed thoroughly and transferred to a sterile 1.5 ml microcentrifuge tube. A transformation mix containing 240 μlpolyethylene glycol(PEG) (50% (w/v)), 36 μl LiOAc(1 M), 25μl ultrapure single-stranded salmon sperm DNA (2 mg/ml) (Clonetech) was added to 50 μl cell suspension. 50 μltransforming DNA (1μg circular plasmid DNA) was added to the above suspension. Whole mixture was vortexed gently and incubated at 30 ̊C for 45 min. 43 μl DMSO was added to the tubeand incubated at 42 ̊C for 15 min. Cells were collected after centrifugation at 5,000 rpm for 1 min and suspended in minimal medium containing 0.6% Bacto-Casamino acid. Transformation mixture was plated on CAA plates and transformants were selected for uracil prototrophy

Yeast transformation usinglithium acetate (LiOAc) strategy

5-10 ml saturated bacterial culture harboring the desired plasmid was harvested at 5,000 g for 3 min. Plasmid DNAwas isolated using QIAprep Spin Miniprep Kit (Qiagen, USA) or GenElute™ HP Plasmid Miniprep kit (Sigma-Aldrich, USA) as per manufacturer’s instructions

Bacterial plasmid isolation

E. coli DH5α ultra-competent cells were transformed with plasmid DNA by heat shock at 42 ̊C for 90 sec as described previously in Molecular Cloning-A Laboratory Manual (Sambrook and Russell,2001). Bacterial transformants were selected on LB agarmediumcontaining appropriate antibiotics. Transformants obtainedwere colony purified on LB plates containing antibiotics.Presence of the desired insertwas first verified by colony PCR followed by PCRusing extracted plasmid DNA as template

Bacterial transformation

suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cellswere immediately snap-frozen in liquid nitrogen and stored at -80 ̊C

A single colony of E.coli DH5-α strain was inoculated in 10ml LB medium and incubated at37 ̊C for overnight. 4 ml of thisovernight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till theOD600reaches to 0.5. Cells were harvested by centrifugation at 2,500 g for 10 min at 4 ̊C and washed gently in 80 ml ice-cold Inoue transformation buffer. Cells were collectedby centrifugation at 2,500 g for 10 min at 4 ̊C and gently resuspended in 20 mlice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. Cell

E. coli DH5α ultra-competent cells preparation

All experiments in this studywere performed with log-phase cellsunless otherwise mentioned. For obtaining log-phase cells, overnight YNB-or YPD medium-grown yeast cellswerere-inoculated in fresh YNB or YPD medium to an initial OD600of 0.1-0.2.Cells were incubated at 30 ̊C with shaking at 200 rpmtill the OD600reached to 0.4-0.6 OD. After incubation, log-phase cellswere collected bycentrifugation at 4,000 rpm for 3 min,washed once with the same medium and usedforfurtheranalysis

Cultivation of logarithmic-phase cell culture

C. glabratastrains were grown overnighteither in YPDor YNBliquid mediumat 30 ̊C with shaking at 200 rpm. Cells were harvested and suspended in 1X PBS to a final OD600of 1.0.Five 10-fold serial dilutions of cell suspension wereprepared in PBS and3-4μlwasspotted on YPD/YNBplates containing various test compoundsusing a multi-channel pipette.Plates were incubated at 30 ̊C and growth profileswererecorded after2-4days

Serial dilution spot assay

Yeast cell viability was measured by plating appropriate dilutions of cell cultureonYPD plates at various time intervalsduringgrowth.Cell suspension was diluted in1X PBS. YPD plates were incubated at 30 ̊C for 2-3 daysand total colony forming units(CFUs)were calculated by counting the number of coloniesthat appeared onYPDplatesand dividing that number by anappropriate dilution factor

Yeast cell viability assessment viacolony forming unit (CFU) assay

preparedin appropriate solvents, sterilizedby autoclaving or filtrationand stored at appropriate temperature

For growth analysisof C. glabratastrains, a single colony from YPD or YNBagar mediumwas inoculated in appropriate liquid medium and incubated at 30 ̊C with shaking at 200 rpmfor 14-16 h. This overnight grown culture was used toinoculatetest medium to an initial OD600of 0.1to 0.3.Optical density/Absorbance of the cell suspensionwas measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at600nmat regular time-intervals up to a period of 96 h.Absorbance values were plotted with respect to time. Generation time of yeast strains wascalculated fromthe logarithmic (log) phase of cellgrowth. Growth profilesbetween 4 (t1)and 8 h(t2)time interval wereconsideredfor calculationof generation time usingfollowing formula. Generationtime(G)= (t2-t1) x {log (2)/ [log (Bf/Bi)]}G= Generation time in ht1=Initial timepoint taken for analysist2 = Final timepoint taken for analysisBf= Number of cells at time t2(calculated on the basis of OD600values, wherein1 OD600of C. glabratacorresponds to 2 X 107cells.)Bi= Number of cells at time t1(calculatedas mentioned above)Severalyeast strains used in this study were analysed for their susceptibility to variouschemical compounds,drugsand metal ions. For this purpose, stock solutions were

Growth assayand measurementof generation time

mM final concentration) and pH was adjustedto the desired valueby addition of HCl or NaOH. Medium was sterilized by autoclaving.YNBagar plates ofdifferent pHwereprepared by mixing equal volume of separately autoclaved 4% bacto-agar solution and2X varied pH-adjusted-YNB liquidmedium.All routine sterilization of mediumand solutionswas either carried outby autoclaving at 121 ̊C for 15-20 minat highpressure condition(15 psi)or filtration with 0.2 μmpolyvinylidene fluoride(PVDF) membranefilter unit (Millex®-GV, Millipore).Both yeast and bacterial strains were stored as frozen 15% glycerol stock at -80 ̊Cfor extendedlifetime

C.glabratastrains were maintainedeither on rich YPDor synthetically-defined YNB medium. C.glabratacells were routinely culturedat 30 ̊Cwith shaking at 200 revolutions per min(rpm)unless otherwise mentioned. Forgrowthexperiments, C. glabratastrains were freshly revived on YPDmediumfrom glycerol stocks.Escherichia coliDH5α bacterial strainwasused for plasmid transformation and propagationpurposes and maintained on LB medium.E.coliBW23473 bacterialstrainwas used to rescue Tn7transposon cassette from C. glabrataTn7insertion mutantsand maintained on LB medium. Bacterial strainsharboring plasmids were maintained on LBagar plates supplemented withappropriate antibiotics.For plasmid isolationpurpose,bacterial strains were grown overnight in liquid LB brothcontainingappropriate antibiotics at 37 ̊C with shaking at 200 rpm. Forpreparation of the solid medium, 2%bacto-agar was added to the mediumand autoclaved. To prepare medium of different pH, YNB mediumwas either buffered with citrateor HEPESbuffer (100

Strainsand culture conditions

Microbiological techniques

Oligonucleotides/primers used in this study were designed using either free online-tool Primer3 (http://frodo.wi.mit.edu/) or Gene Runner software (http://www.generunner.net/). Oligonucleotides used in this study were commercially

synthesized from MWG Biotech Pvt. Ltd., Bangalore. All primers used in this study are listed in Table 2.3.Table 2.3: List of primers used in this study

Oligonucleotides

10mM EDTA0.1% SDS 1 M ureaToluidine blue staining solution:0.05% Toluidine blue20% Methanol2% GlycerolSolution was prepared in H2O.Destaining solution for polyphosphate gels:20% Methanol2% GlycerolSolution was prepared in H2O.Spheroplast buffer:50 mM Potassium phosphate (pH 7.5)0.6M Sorbitol0.2 X YPD mediumPS(PIPES-Sorbitol)buffer:10 mM PIPES-KOH (pH 6.8)200mM Sorbitol1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Citric-Phosphate buffer:0.5 M citric acid0.5 M dibasic sodium phosphatepH was adjusted to 5.0 with phosphoric acid and filter-sterilized.MES/TEA buffer:1 mM MES(2-(N-morpholino)ethanesulfonic acid)pH was adjusted to pH 5.0 with TEA(triethanolamine).Plasma membrane suspension buffer:50 mM Tris-HCl(pH 7.5)0.1mM EDTA0.1 mM Dithiothreitol 20% GlycerolPolyphosphate extraction buffer:50 mM HEPES (pH 7.2)

Genomic DNAisolation buffersBuffer A:50 mM Tris-HCl10mM EDTA150 mM NaCl 1% Triton-X 1% SDSBuffer B:50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol(To be added just before use

Other buffers

15% Acetic acidTris-Borate Saline(TBS):25 mM Tris150 mM NaClpH was adjusted to 7.4withHCl.This was prepared as 10 X stock solution and used at 1 X concentration.Blocking and wash buffers(PBS-T and TBS-T):5% Fat-free milk 0.1% Tween-20 Volume was made to 100 ml either with 1 X PBS(PBS-T)or 1 X TBS(TBS-T)

0.02% Bromophenol blue 2% DTT This was prepared as a 4 X stock solution and used at a 1 X concentration.SDS-PAGE running buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS This was preparedas a 10 X stock solution and used at a 1 X concentration.Coomassie brilliant blue (CBB) staining solution:50% Methanol10% Acetic acid0.1% Coomassie brilliant blue-R250Western blotTransfer buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS Thiswas preparedas a 10 X stock solution and used ata 1 X concentration.1X Transfer buffer (1litre):200 ml of methanol 100 ml of 10 X transfer buffer 700ml of waterPonceau 3S staining solution:0.25% Ponceau 3S40% Methanol

SDS-PAGE30% Acrylamidesolution29 g Acrylamide1 g Bis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS):10 g SDS in 100 mlH2OResolving gel mix (12%) (15 ml): 4.89 ml H2O6 ml 30% acrylamide:bisacrylamide (29:1) mix3.8 ml 1.5 M Tris-HCl (pH 8.8) 150 μl 10% SDS 150 μl 10% APS 10 μl TEMEDStacking gel mix (3 ml):1.689 ml H2O500 μl 30% acrylamide:bisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8) 30 μl 10% SDS 30 μl 10% APS 10 μl TEMEDSDS loading buffer:130 mM Tris-HCl (pH 8.0) 20% (v/v)Glycerol 4.6% (w/v) SDS

Whole cell lysis buffer(Homogenizing buffer):50 mM Tris-HCl(pH 7.5)2 mM EDTA10 mM sodium fluoride*1 mM sodium orthovanadate*1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Protein isolation and SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)

Protein isolation and SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)

Buffer C:100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSRNA isolation bufferAE buffer: 3 M Sodium acetate0.5 M EDTA(pH 8.0)Phenol:Chloroform:Isoamyl Alcohol (25:24:1)solution:25 volume of Phenol24 volume of Chloroform1 volume of Isoamyl alcholDNA sampleloading buffer:0.25% Bromophenol blue0.25% Xylene cyanol15% Ficoll

Genomic DNA and RNA isolation buffers

15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O pH was adjusted to 6.7 with 1 N KOH. MnCl2needsto beaddedseparately,drop by drop with stirring, tothe buffer. PIPES goes into solutionwhenpH is greater than 6.7. The solution, after pH adjustment to 6.7 was filter-sterilized and stored at -20ºC.Reagents for yeast transformation:1 M Lithium acetate (LiOAc)50% Polyethylene glycol10 mg/ml Carrier DNADimethylsulfoxide (DMSO)

INOUE transformation buffer:For bacterial DH5α ultra-competent cells preparation10 mM PIPES (free acid)

Transformation-related solutions

10 mM Tris-HCl (pH 8.0)1 mM EDTA Tris-Acetic acid EDTA (TAE) buffer:40 mM Tris base 0.5 M EDTApH was adjusted to 8.5 with glacial acetic acid.This was prepared as a 50 X stock solution and used at a 1 X concentration. Tris-Borate EDTA (TBE) buffer:90 mM Tris-borate 2 mM EDTA (pH 8.0) pH was adjusted to 8.3withHCl.This was prepared as a 10 X stock solution and used at a 1 X concentration.Both TAE and TBE were used asstandard gel electrophoresis buffers.HEPES buffer:This was used to prepare YNB medium of different pH.1M HEPESpH was adjusted to 7.5withNaOH.Bufferwas filter-sterilized and stored in an amber-coloured bottle. Citrate buffer(0.1M, pH 5.5):4.7 volume of 0.1 M Citric acid 15.4 volume of 0.1 M Sodium citrate

Phosphate-Buffered Saline (PBS):137 mM NaCl 2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-HCl buffer:0.5 M TrizmaBase pH was adjusted to7.6 using concentrated HCl.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-EDTA (TE)buffer:

Common buffers

Buffersand solutions

0.67% Yeast Nitrogen Base2% DextroseYeast Carbon Base (YCB):1.17% Yeast CarbonBase1% DextroseCAA:0.67% Yeast Nitrogen Base 2% Dextrose0.6% Casamino acids Plates weremade by adding 2% agar

Yeast Extract-Peptone-Dextrose (YPD):1% Yeast extract2% Peptone 2% DextroseYeast Nitrogen Base (YNB)

Yeast medium

Luria Bertani (LB):0.5% Yeast Extract1% Tryptone 1% NaCl LB-ampicillinand LB-kanamycin plates:LB medium50 μg/ml ampicillin30 μg/ml kanamycinSuper Optimal Broth (SOB): 0.5% Yeast extract2% Peptone 10 mM NaCl2.5 mM KCl10 mM MgCl210 mM MgSO4

Bacterial medium

antibodies,anti-mouse IgG andanti-rabbit IgG conjugated with horseradish peroxidase (HRP) were obtained from Cell Signaling Technology, USA

All chemicals were purchasedfrom commercial sources. Mediacomponents for bacterial and yeast growthwere obtained from BD (Becton, Dickinson and Company, USA). Other chemicals were purchased from Sigma-Aldrich Co., USA. Materials used in recombinant DNA experiments were primarily obtained from New England Biolabs, Invitrogen, Bangalore Genei and MBI Fermentas. SuperScript™ III first-strand synthesis system was purchased from Invitrogen.MESA GREEN qPCR MasterMix Plus for SYBR®Assay was purchased from Eurogenetec. Kits used for plasmid isolation, PCR product purificationand DNAgelextractionwerefrom Qiagen.Radioactive chemical, ortho-P32-phosphoric acid,wasprocured from BRIT-Jonaki, CCMB, Hyderabad.Anti-Pma1 polyclonal antibody raised against S. cerevisiaePma1 was purchased from Santa CruzInc.,USA. Anti-phospho-p44/42 MAPK (Thr202/Tyr204) was purchased from Cell Signaling Technology, USA. Anti-CPY polyclonal antibody raised against S. cerevisiaeCPY was procuredfrom Thermo Scientific. Anti-Gapdh antibody raised against human Gapdh was purchased from Abcam. Secondary

Chemicals and antibodies

All C. glabratastrains and plasmids used in this study are listed in Tables 2.1 and 2.2, respectively.Table 2.1: List of yeast and bacterial strains used in this study

Strains and plasmids

Centre for DNA Fingerprinting and DiagnosticsHyderabad, India

Gaurav Bairwa

Studies on virulence factors of Candida glabrata

activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner

To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the

Seperation of phospholipids by thin layer chromatography(TLC)

PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform

PI-3 kinase reaction set up and phopsholipid extraction

10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice

Preparation and sonication of phosphatidylinositol-sodium salt solution

A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use

Preparation of cell lysate

In vitroPI-3 kinase reactions wereset up to measure PI-3P synthesized as described earlier(Whitman et al., 1988)

Phosphatidyl inositol-3 kinase (PI-3 kinase assay)

Colony blot assay was performed to analyse secretion of carboxypeptidase Y(CPY)as described previously (Roberts et al., 1991). Single colony of a C. glabratastrain was inoculated in YPD medium andculture was grown till stationary phase. 0.1 OD600equivalent cellsfrom this culture were spotted on CAA medium,overlaidwith a nitrocellulose membrane and plate was incubated at30 ̊C for 18-20 h.Afterincubation, nitrocellulose membranewas washed with water to remove cells and membrane-bound CPYwas detected by immunoblotting with polyclonal anti-CPY antibody at a dilution of 1:10,000

Colonyblot assay

was washed three times with PBS to remove non-adherantC. glabratacellsand Lec-2 cells were lysed in 5% SDS. Lysates were transferred totubes containing scintillation fluidand radioactive counts obtained were considered as ‘output values’. Percentage adherence wasdetermined using following formula

Adherence of C. glabratacells toLec-2 epithelial cells wasmeasured as described previously(Cormack et al., 1999).Lec2cells were seeded ina 24-well tissue culture plate at a seeding density of 5X105cells per well and allowed to adhere for 12 h. After 12 h,medium supernatant was discarded by inverting the plate in a reservoir and cells were washed thrice with PBS. Lec2 cells were fixed in 3.7% para-formaldehyde for 15 minfollowed by 2 PBS washes. PBS containing antibiotics, penicillin and streptomycin,was added toeach well of the 24-well plate and Lec-2 cellswere stored at 4°C.For adherence measurement,strains were taken out either on YPD or CAA mediumandgrown at 30°C for 2 days. Single colony of a C. glabratastrain wasinoculated in 10 ml CAA medium ina 100 ml culture flaskand allowed to grow at 30°C for 16-20 h. 100 μlyeast culture wasreinoculated in fresh 5 ml CAA liquid medium in a 15 ml polypropylene tube. 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN labelmix(JONAKI, India) was added to thetube and cultures were grown at 30°C for 16-20 h for radiolabeling of C. glabratacells. C. glabratacells from 1 ml culture were harvested and washed threetimes with PBS to remove residual S35(Met:Cys-65:25) labeling mix from medium supernatant. Next,cells were resuspended in 1 ml PBS. OD600was measured and cell suspensions of 0.5 OD600were prepared. PBS was aspirated out of the wells of 24-well plate containing fixed Lec-2 cells. 200 μl of S35(Met:Cys-65:25)-labeled C. glabratacell suspensions were added to each well. To determine the total amount of radioactivity present in labeled C. glabratacell suspension, 200 μl of S35(Met:Cys-65:25)-labeledC. glabratacell suspensions were transferred to a scintillation vial containing scintillation fluid. Radioactive counts present in this fraction were considered as ‘input values’. For measurement of yeast adherence to Lec-2 cells, plates were centrifuged at 1,000g for 5 min and incubated for 30 min at room temperature. Following incubation, each wel

Adherence assay

20 mg protein samples, isolated from RPMI-grown and macrophage-internalized yeast, were usedto measure KDAC activityusing HDAC Fluorimetric Assay/Drug Discovery Kit (EnzoLifeScience) as per manufacturer’s instructions

Lysine deacetylase (KDAC) activity measurement

For protein extraction, yeast cells were suspended in 50-100μl protein extraction buffer containing 320 mM (NH4)2SO4, 200 mM Tris-Cl (pH 8), 20 mM EDTA (pH 8), 10 mM EGTA (pH 8), 5 mM MgCl2, 1 mMDTT, 10% glycerol and protease inhibitorsand disrupted using glass beads.Cell lysate was centrifuged at 7,500g and4oC for 15 min. 30 μg of total protein was resolved on a 15% SDS-PAGE gelat 32 mA till the dye front reachedthe bottom. Resolved proteins were transferred to Hybond-P membrane at 350 mA for 1.5 h in the cold room.Transfer of the proteins was visually confirmed by examining marker’s lane and membranes wereincubated in a small box for 2 h in 5% fat free milkprepared in 1X TBST for blocking. Blocking solutions were discarded and primary antibody, appropriately diluted in 5% fat free milkprepared in 1X TBST,was added to the box containing membrane. After overnight incubation in primary antibody, membranes were washed thrice with 1X TBST for 10 min. Membranes wereincubated for 2 h inappropriate secondary antibodydiluted in 5% fat free milkprepared in 1X TBST. Blots were either developedby chemiluminescence based ECL-Plus western detection system orChemidocTMgel imagingsystem. CgGapdhwas used as a loading control. To exclude the possibility of any contribution of THP-1 proteins tocell extracts prepared frommacrophage-internalized yeast, two control experiments wereperformed. First, we probedthe blots with antibodies specific for mammalian tubulin and actin.As expected, we neitherdetectedanysignal for mammalian actin nor formammalian tubulin. In the second control experiment, we treated macrophage lysates with proteinase-K prior to the yeast pellet disruptionand probed yeast lysates for different histone modifications.This proteinase-K treatmentdid not alter the epigenetic signature of C. glabratacells.Together, these data indicate that yeast protein samples were devoid ofany mammalian protein contamination

Protein extraction and immunoblotting

homogenizedin 1 ml PBS and fungal burden was assessed by plating appropriate dilutions of tissue homogenate on YPD plates containing penicillin and streptomycinantibiotics (100units/mlpenicillin and 100μg/mlstreptomycin). All mice experiments were repeated twice with a set of 7-8 mice per strain in each experiment

Experiments involving mice were conducted at VIMTA Labs, Hyderabad.100 l YPD-grown C.glabratacellsuspension(4 X 107cells)was injected into female BALB/c mice (6-8 weeks old) through tail vein. Seven dayspost infection, mice weresacrificedand kidneys, liver,spleenand brainwere harvested. Organs were

Mouse infection assay

Experiments involving mice were conducted at VIMTA Labs Limited, Hyderabad in strict accordance withguidelines of The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/OS/05). Procedures used in this protocol were designed to minimizeanimalsuffering

Ethics statement

Othermethods

CgRTT107(3.3 kb),CgRTT109(1.3 kb),CgVPS15(3.4kb) and CgVPS34(2.4kb) ORFs were PCR amplified from genomic DNA of the wild-type strain using high fidelity Platinum Pfx DNA polymerase with primers carrying restriction sites for SmaI-SalI,BamHI-SalI,XmaI-XhoIandSalI-XmaI,respectively.Amplified fragments werecloneddownstream of the PGK1promoterin the pGRB2.2 plasmid. Clones were verified by bacterial colony PCR, sequencing and complementation analysis

Cloningof C. glabrataORFs

After restriction enzyme digestion, digested products wereresolved on agarose gels and desired DNA fragmentswereextracted from the gel. Concentration of gel-extracted DNA fragments was determined usingspectrophotometerand ligation reactions were set up using a molar ratio of vector to insert of 1:3 and 1:1 for sticky and blunt end ligations, respectively. Ligation mixwas incubatedeither at 22ºC for 4 hor at 16°Cfor 14-16 h. After incubation,T4DNA ligase was inactivatedat 65ºC for 20 min

Ligation

QIAGEN QIAquick PCR purification kit containing buffers, spin columns and collection tubes wasused topurify DNA fragments from PCR andenzymatic digestion reactions as per the kit manufacturer’s instructions

Purification of restriction enzyme-digestedand PCR amplifiedproducts

QIAGEN QIAquick Gel extraction kit containing required buffers, spin columns and collection tubes was used to extract and purify DNA from agarose gels. Digested DNA sample was resolved on 1-1.2% agarose gel and gel piece containing desired fragment was cut ona UV-transilluminator. DNA fragment was purified as per the kit manufacturer’s instructions

Gel extraction of DNA

C. glabratacells grown either in RPMI medium or harvested from THP-1 macrophages were collected, washed with DEPC treated water and were disrupted with glass beads in trizol. Total RNA was isolated using acid phenol extraction method and frozen at -80C. Quality of RNA was examined by determiningtheRNAintegrity number (RIN) before microarrayanalysis.Microarray experiments wereperformed atOcimum Biosolutions Ltd., Hyderabad (http://www.ocimumbio.com). Briefly, a4x44K GE Agilent array comprised of 10,408 probes representing 5,205 ORFs of C. glabratawas used wherein average number of replicates for each probe was four to five. Feature Extraction software version 10.7.3.1. (Agilent) and Quantile normalization was used for data analysis. Hierarchical clustering was performed using Complete Linkage methodwith Euclidean Distance as distance measure. Data arethe average of two hybridizations from biological replicates ofeach sample and raw data sets for this study areavailable at the Gene Expression Omnibus database(Accession number -GSE38953)

Microarray Analysis

To preclude the possibility of human RNA contamination, cDNA prepared from internalized yeast was examined for the presence of human transcripts encoding Ccl5 and histone H3. However,no amplification forhuman genes was observed, thus, eliminating any possiblecontamination of THP-1 RNA with yeast RNA

Primersfor real-timePCR analysisweredesigned using Primer3 plus software and are listed in Table 4. To extractRNA from macrophage-ingested C. glabratacells, infected THP-1cells were washed twice with PBS and lysed in 1 ml ice-cold water. Lysate was centrifuged followed by two quick washes with DEPC-treated water andwashed yeast cell pellets were frozen on dry ice.For RNA extraction, yeast cells were disrupted with glass beads in trizol and total RNAwas isolated usingacid phenol extraction method described above.Optimal primer and cDNA concentrations were standardizedand qRT-PCR was performedusing ABI 7500 Fast Real-Time PCR System (Applied Biosystems).In brief, 0.5 μl cDNA,0.1 to 0.2picomoles of gene specific primers and 10 μl 2X MESA GREENqPCR™ Mastermix Plus containing SYBR green dye (Eurogentec)were mixed in thewellsof a 96-well PCRplate (Axygen). Final reaction volume was adjusted to 20 μl with DEPC-treated water. Transcript levels were quantified with an end-point value known as Ct(cyclethreshold). Expression of TDH3, which encodes CgGapdh,was used asaninternal control. The Ct defines the number of PCR cycles required for the fluorescent signalof SYBR green dye to cross beyondthe background level.Fold-change in transcript expression was determined usingfollowing formula.Fold change in expression = 2-ΔΔCtΔΔCt= ΔCttreated -ΔCtuntreatedΔCttreated = Ctvalue forthe gene of interest under treated condition -Ctvalue forthe internal control gene (TDH3) under treated conditionΔCt untreated = Ctvalue forthegene of interest under untreated condition -Ctvalue forthe internal control (TDH3) gene under untreated condition

Quantitative real-timePCR

Complementary-DNA synthesis was doneusing reverse transcriptase enzyme and oligo-dT primers. For this, 1 μg good quality RNA was treated with1μl(1 unit) DNase I for 15 min to remove DNA contamination. Next, SuperScript III First-Strand Synthesis System kit (Invitrogen) was used to synthesize cDNA according to the manufacturer’s instructions. cDNA synthesized was stored at -20 ̊C till further use

Synthesis of complementary DNA (cDNA)

For RNA experiments, all solutions were prepared in RNase free diethylpyrocarbonate (DEPC) water. Microcentrifuge tubes and tips used for RNAworkwere autoclaved twice and driedat 70 ̊C for overnight before use. Non-autoclavable plastic items were wiped with Ambion RNAseZap to remove RNAse contamination, if any. RNA was extracted from C.glabrata cells usingacid phenol extraction method. C. glabratacells were harvested at 2,500g for 5 minat 4 ̊C, resuspended in 1 ml ice-cold DEPC water and were transferred toa2 ml microcentrifuge tube. Cells were spun down at 6,000g for 3 minat 4 ̊C and resuspended in 350 μl AE solution. Next, 50 μl SDS and 400 μl acid phenol(pH 4.5)solutions were added to thetube and mixed well by vortexing. The tube wasincubated at 65 ̊C for 15 min with continuous mixing. After incubation, tube was kept on ice for 5 minand centrifuged at 7,500g for 5 minat 4 ̊C. Aqueous phase was transferred toa new 1.5 ml microcentrifuge tube and RNAwasextracted with an equal volume of chloroform. Total RNA was precipitated at room temperature with 1/10thvolume of 3 M sodium acetate (pH 5.2) and 2.5 volumesof chilledabsolute ethanol for 20 min. Precipitated RNA was collected by centrifugation at 7,500g for 5 minat 4 ̊C. RNA pellet was washed with chilled 70% ethanol and resuspended in 50 μl nuclease-free water. RNA concentration was determined by measuring absorbance at 260 nm. Quality of extracted RNA was examined by gel electrophoresis on 0.8% agarose gel prepared in DEPC-treated TAE buffer

RNA extraction

Nucleosomal-associated DNA was extracted from RPMI-grownand macrophage-internalized C. glabratacells using EZ NucleosomalDNA prep kit (ZYMO Research),treated withmicrococcal nuclease digestionfor 2.5, 5, 7.5 and 10 min at 25ºC andwasresolved on 2% agarose gel

Micrococcal nuclease digestion assay

temperature. Genomic DNA pellet was dissolvedeitherin 50 μl 0.1X TE or molecular biology grade water containing 0.3 μlAmbion RNAse cocktail and incubated at 37ºC for 30 min.After RNA digestion, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis

Desired C. glabratastrain wasgrownovernight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500g in 15 ml polypropylene tube.Yeast cells were washed with PBS, resuspendedin 500μl lysis buffer (Buffer A) andwere transferred toa2ml microcentrifuge tube. Yeast cells were incubated for 15 minon a thermomixer set at 65 ̊C and 750 rpm. After incubation, 0.5 gm glass beads (0.5 mm) and 500 μl PCI solution were added to thetube. Yeast cells were lysedthree times for 45 seconds each on a bead beating apparatus with intermittent cooling on ice to prevent overheating. Cell lysates were centrifugedat 7,500gfor 5 minandupperaqueous phase (300-350 μl) wastransferred carefully to a new 1.5 ml microcentrifuge tube. 1 mlabsolute ethanol was added andmixedwellby inverting the tube3-4 times. To precipitate genomic DNA, suspension was centrifuged at 7,500g for 10min.Precipitated genomic DNA was washedwith 70% ethanolanddried at room

Genomic DNA isolationby glass bead lysis method

C. glabrata cells were grown overnight in 10 ml YPD liquid medium. Cells were harvested at 2,500g for 5 min, resuspended in 400 μl Buffer A (50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 1% SDS) and were transferred to a 2 ml microcentrifuge tube. Equal volume of phenol-chloroform-isoamyl alcohol (PCI) solution was added tocell suspension and tubes were vortexed for 2-3 min. After incubation at 42 ̊C for 30 minon a thermomixer set at 800 rpm (Eppendorf), cell debris was removed by centrifugation at 7,500g for 10 minand aqueous phase (300-350 μl) was carefully transferred to a new 2 ml microcentrifuge tube. Genomic DNA was precipitated with800 μl chilled absolute ethanol and 35 μl sodium acetate (3 M, pH 5.2). DNA pellet was washed with chilled 70% ethanol and dried at room temperature for 5-10 min. Genomic DNA pellet was dissolved either in 50 μl 0.1X TE or molecular biology grade water containing 0.3 μl Ambion RNase cocktail and incubated at 37 ̊C for 30 minfor digestionof RNA. After RNA degradation, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis

Genomic DNA isolationby quick genomic DNA extraction method

Stripping of membranes in buffer containing 0.4 M NaCl yielded slightly better results. Hybond membranes were reused for 5-10 times after stripping

Radiolabeled-bound probes were stripped from the membrane by boiling in 1% SDS containing 0.1X SSC for 15 min. Alternatively, membraneswereincubatedtwicein stripping solution (0.4 M NaOH)at 45°C for 30 minto remove the bound probes

Stripping of probes from hybridized membranes

After 2-3 h exposure,phosphorimagerscreenwas scannedon Fuji FLA-9000 to acquire hybridization images. Next,signal intensity for each spot on the membrane for both input and outputsampleswas quantifiedusing Fuji Multi Gauge V3.0 software andpercentage intensity foreach spot relative tothe whole signal intensity ofthe membranewas determined.To identify mutants with altered survival profiles,ratio of output (Op) to input (Ip) signal for each spot (oligonucleotide tag)present on the membranewas calculated.Mutantsdisplaying at least 6-fold higher and 10-fold lower survival were selectedas “up’ (Op/Ip= 6.0) and ‘down’ (Op/Ip = 0.1) mutants, respectively

Data analysis

After14-16 hincubation, hybridization buffer was decanted to a radioactive liquid waste container.Membraneswere washedtwice with 2X SSC (saline-sodium citrate) containing 0.1% SDS for 15 min at 55°C followed by two washes with 1X SSC containing 0.1% SDS for 15 min at room temperature. Post washes,membranes were rinsed with 1XSSC buffer at room temperature and exposed to phosphorimager screen for 2-3 h

Post-hybridization washes

Primers (OgRK 25 and OgRK 26, 200 pM ) –1 μl, eachTaq DNA polymerase –0.5 μlαP32-dCTP( specific activity-3,000 Ci/mMol)–2.5 μlWater –38.5 μlαP32-dCTPlabeled PCR productswere denatured by incubatingtubesat100°C for 5 min followed by immediatechilling on ice. After 2 h prehybridization, radiolabeleddenatured input and output probes were added to respective bottles and bottles were transferred to a hybridization oven set at 42ºC and 6 rpm

Two Hybond-N membranes,each carrying 96 immobilized DNA with unique signature sequences,were transferred to 15 cm long hybridization bottlesandlabeledas input and output. 10 mlprehybridization buffer was added to each bottle and bottles were transferred to a hybridization oven setat 42ºC and 6 rpm. To prepare input and output probes,genomic DNAsfrom input and output C. glabratacell pelletswere extracted using glass bead lysismethodandunique signature tags were PCR amplifiedwith nucleotide mix containing αP32-dCTP using primerscomplementary to the invariant region flanking each unique oligonucleotide sequence.Following is the composition of50 μlPCR cocktail used to prepare radiolabeledinput and output probes.10X PCR buffer–5 μldNTP mix (without dCTP, 2mM) –0.5 μldCTP (0.05 mM) –1 μl

Southern hybridization

E. colistrains containing plasmids with unique oligonucleotide signature sequences were inoculated in LBmedium containing ampicillin and grown overnight at 37°Cand 200 rpm. Plasmids were extracted, quantitated anddenatured in alkaline denaturing solution. Approximately, 200 ng of each plasmid DNA was transferred to theHybond-Nmembraneusing96-well Dot Blot apparatus. Membranes were neutralized in 2X SSC and denatured plasmids were cross-linked to Hybond-N membranes usingUV cross linker

Membrane preparation

E. colistrains carrying plasmids were inoculated and grown overnight at 37ºC and 200 rpm in LB-liquid medium supplemented with either 50 μg/ml ampicillinor 30 μg/ml kanamycin. Cells were harvested by centrifugation at 2,500g for 5 min. Plasmids were extracted using Qiagen plasmid miniprep kit following the manufacturer’s instructions. Concentration of the extracted plasmid DNAs was measured using spectrophotometerat 280 nmandstored at -20ºC

Plasmid DNA purification

Molecular biology methods

Genomic mapping of disrupted locusin Tn7insertion mutants was carried out as describedpreviously(Kaur et al., 2004).C. glabratamutants carrying Tn7insertionswere grown in YPD-liquid medium and genomic DNA was isolated fromovernight cultures. 10 μg genomic DNA was digested either with restriction enzyme MfeIor SpeI.Restriction enzyme-digestedDNA was precipitated with 1 ml ethanol and 1/10thvolume of sodiumacetate (3 M,pH 5.2). DNA pellet was washed twice with ice-cold 70% ethanol, air driedand was resuspended in sterilewater. DNA was recircularized with T4 DNA ligase.Resultant circular DNA carriedTn7cassette flanked on bothsidesby the disrupted locus oftheC. glabratagenome. CircularDNA wastransformed in E. coliBW23473 strainwhich contains protein Π (the product of the pirgene) required by R6Kγorifor replication.Twoverified transformants were grown overnight in LB-kanamycin medium and plasmids were extracted. Purified plasmids were sequenced withprimers reading outwards (OgRK 183 and OgRK 184) from both ends ofTn7cassette.Sequences obtained were compared,usingBLAST,against C. glabratagenome sequence database and regionsof Tn7insertions in C. glabratawere mapped

Mutant rescue

E. coli BW23473 electro-competent cell aliquots were taken out from -70ºC freezer, thawed on ice and were mixed with 1-2 lplasmid DNA. Mixture was pulsed with the Gene Pulser® electroporation apparatus (Bio-Rad),set at 1800 Volts, 25 μF and 200 Ω,in a chilled 0.1 cm electroporation cuvette. After electric pulse, 1 ml LB medium was immediately added to the cuvette and suspension was transferred to a 1.5 ml sterile microcentrifuge tube. Cells were incubatedat 37°C and 200 rpm for 1 h, centrifuged and were plated on LB-agar plates containing kanamycin (30 μg/ml). Transformants were colony purifiedon LB-kanamycin plates. Positive clones were verified by colony PCR and inoculated in LB-liquid medium containing kanamycin (30 μg/ml) for plasmid isolation

Transformation of E. coliBW23473 cells by electroporation

To prepareelectrocompetent cells, a single colony of E. coli BW23473 strain from a freshly-streaked LB agar plate was inoculated in 50 ml LB liquid medium. Culture was incubated at 37°C for 14 h with continuous shaking at 200 rpm. 25 mlovernight-grown E. coliBW23473 culture was transferred to 500 ml LB liquid medium and incubated at 37°C till the OD600 reached to 0.4. Post incubation, cultures were transferredto ice and centrifuged at 1,000g for 15 minat 4°C. Cells were washed twice with 500 ml ice-cold sterile water, three times with 250 ml ice-cold 10% glycerol solution and resuspended in 1 ml 10% glycerol solution. After absorbance measurement, cell suspension was normalized to final cell density of 3X1010cells/ml and dispensed in 50 μl aliquots to sterile ice-cold 1.5 ml microcentrifuge tubes. Aliquots were snap frozen in liquid nitrogen and stored at -70ºC

Preparation of E. coliBW23473electrocompetent cells

E. coliDH5α strain was transformed with plasmids carrying appropriate inserts to clone and generatedeletion strains of C. glabrataORFs(Sambrook, 2001). Ultracompetentcells stored at -70⁰C were thawed on icefor 5-10 min. 5 μlligated plasmid was added to100 μlultracompetent cells andcells were incubatedon ice. After 30 min, competent cells were subjected to heat shock at 42⁰C for 90 seconds. Cells were immediately transferredtoicefor 2-3min. Next, 800 μlSOC (or LB) medium was added and cells were allowed to recover for 45 minon a shaker incubator set at 37⁰C.After the recovery, cells were centrifuged at 2,500g for 4 min. Medium supernatant was discarded and cells were resuspended in 200 μlfresh sterile LBmedium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates wereincubatedat37⁰C for 12-16 h

Bacterial transformation

10 min at 4 ̊C and gently resuspended in 20 ml ice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. Cell suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80 ̊C

A single colony of E. coli DH5α strain was inoculated in 10 ml LB medium and incubated at 37 ̊C for overnight. 4 ml overnight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till the OD600 reached to 0.5. Cultures were centrifugedat 2,500 g for 10 min at 4 ̊C and harvested cells were washed gently with80 ml ice-cold Inoue transformation buffer. Cells were collected by centrifugation at 2,500 g for

Preparation ofE. coli DH5α ultracompetent cells

A microtipful of cellsfor each yeaststrainfrom appropriate mediumwassuspended in 10μlzymolyase cocktailandincubated at 37ºC for 90 min. 2 μlof zymolyase-treated cell suspension was used as template in 25 μlPCR reaction

Yeast colony PCR

Alipophilic styryl dye,FM4-64,is a vital stain which istakenupby cells viaendocytosis through plasma membrane(Vida and Emr, 1995). Therefore, it fluorescesonly in live cells. Importantly, neitherfixed cells canbe stained with FM 4-64norcells canbe fixed afterFM 4-64staining. For vacuole staining, single colony of the test strain grown onYPD plate was inoculated in 10 ml YPD medium for overnight. 100 μlovernight culture was inoculated in fresh YPD medium and incubated at 30ºC for 3 hto obtain log-phase cells. C. glabratacells from 1 ml log-phase culture were harvested at 4,000 rpm for 5 minin a table top centrifuge. Supernatant was aspirated out,cells were resuspended in 50 μl YPD medium and 1 μl FM 4-64 (16 μM final concentration) was added.C. glabratacells were incubated in a 30ºC water bath for 30 min. 1 mlYPD medium was added and cells were harvested at 4,000 rpm for 5 minin a table-top centrifuge. After discarding supernatant,C. glabratacells were washed with fresh YPD medium and resuspended in 1 ml YPD medium. C. glabratacells were incubated at 30ºC for 90 min, washed with 1 mlsterile water and were resuspended in 50 μl YNB medium. Labeled C. glabratacells were observed underfluorescence microscope in red filter(730nm)

Stainingof C. glabratavacuoleswith FM4-64

centrifugation at 5,000 rpm for 4 minat room temperature. Harvested cells werewashed with PBS and treated with different compoundse.g.H2O2. After treatment,cells were harvested and further processed according to the type of experiments performed

For several experiments, log-phase C. glabratacells were harvested and treated with different compounds. For this, single colony of aC. glabratastrain was inoculated in YPD-liquid medium and grown for 14-16 h at 30ºC withcontinuous shaking at 200 rpm. Overnight cultures were reinoculated in YPD medium to an initial OD600of 0.1 andgrown for another 4 h. These log-phase cells were harvested by

Harvesting of and treatment to logarithmic phase C. glabratacells

For opsonization,C. glabratacells were incubatedwith 1 μg/μl human IgG for 30 min at 37°C and washed thrice with PBS. Alternatively, yeast cells were incubated with 25% human serum at 37°C for 30 min followed by threePBS washes

Opsonizationof C. glabratacells

C. glabratastrains were grown overnight in YPD medium. Cellswereharvested from 1 mlcultureandwashed withPBS.Cells were next washed with50mM NaH2PO4andresuspendedin 100 μlFITC-dextran(50mg/ml). After incubation at 37°Cfor 45 min, cells were washed thrice with PBS for complete removal of FITC-dextran.Yeast cells were resuspended in 1 ml PBS and used to infect PMA-treated THP-1 cells in 4-chambered glass slide

Fluorescein isothiocyanate(FITC)staining of C. glabratacells

24 h post infection, THP-1 macrophages were washed thrice with PBS, lysed in water and recovered yeast cells were used to infect THP-1 cells at a MOIof 1:10. Three rounds of macrophage infection foreach mutant pool were carried out to enrich for the desired mutants in the final population. The lysate of 3rdround infection was inoculated in YPD medium for overnight (output). Cells were harvested, genomic DNA isolated from each input and output cell pellet andunique signature tags were PCR-amplified with P32-labeledα-dCTP using primers complementary to theinvariant region flanking each unique tag sequence. LabeledPCR products were denatured at 95°C for 10 min, chilled on ice and were hybridized tonylon membranescarrying immobilized plasmid DNA containing 96 unique tagsfor 14-16 h at 42°C.Membranes were washed twicewith 0.1X SSC bufferand exposed to phosphorimager screen for 2-4 h. Radioactive counts for each spot were quantified using Image Quant and Fuji Multi Gauge V3.0 software. Relative percentage intensity for individual spot was calculated with respect to allspots present oneach hybridizedmembrane

YPD-grown cultures (0.05 OD600) of each mutant pool (96 mutants, each carrying a unique signature tag) were either inoculated in YPD medium for overnight (input) or used to infect differentiated THP-1 cells (1X106). After 2 h incubation, non-cell-associated yeastcellswere removed by washing THP-1 cellsthricewith PBS. At

Screening of C. glabrataTn7insertion mutant library

Single colony of C. glabratastrains wasinoculated in 10ml YPD-liquid medium and grown at 30°C with constant shaking at 200 rpm for 14-16 h. Overnight culture was used to inoculate 10 ml YPD broth to an initial OD600of 0.1 and culture was grown for 4-5 h to obtain log-phase culture. Log-phase C. glabratacells were harvested in 15 ml sterile polypropylene tubesby centrifugation at 4,000 rpm for 5 min. Harvested cells were washed with10ml sterile water,resuspendedin 1 ml sterile water and transferred to a 1.5 ml microcentrifuge tube. Cells were harvested at 4,000 rpm for 5 minand resuspended in 100 μl of100mM lithium acetate solution.Yeast transformation cocktail was prepared in a 1.5 ml microcentrifuge tube by mixing 240 μlpolyethylene glycol(50%), 36μl lithium acetate(1 M) and25μlheat-denatured single stranded carrier DNA(2 mg/ml). 50 μlC. glabratacell suspension and 50 μltransforming DNAwas added to the transformation cocktail, mixed well andincubatedat 30 ̊C for45 min. 43 μlDMSO was added and cells were subjected to heat shock at 42 ̊Cfor 15 min. After the heat shock, cells were transferred to ice for 10-15 seconds, centrifuged at 4,000 rpm for 5 min and supernatantwas removed.Cells wereresuspended in 200 μlsterile water andspread platedonappropriate selectionmedium. Plates wereincubatedat30 ̊Cfor 2-3 days

Yeast transformation

Identified mutants were phenotypically characterized in 96-well plate format. Mutant cultures were grown in YPD medium for overnight, diluted 150-fold in PBS and 5 μl of cell suspension was spotted on different plates with a 96-pin replicator. Growth was recorded after 1-2 daysof incubation at 30°C

Phenotypic profiling

at 30°C andimages were captured after 2-8daysof incubationdepending upon the medium used

Yeast strains were grown in YPD medium for 14-16 hat 30°Cunder continuous shaking at 200 rpm. Cells were harvested from 1 mlculture, washed with PBS and were diluted to an OD600of 1. Five ten-fold serial dilutions were preparedfrom aninitial culture of 1OD600.4 μl cultureof each dilution was spotted onYNB-agar plates containing different carbon sources. For spotting on YPD plates containing different compounds, 3 μl cultureof each dilution was spotted. Plates were incubated

Serial dilution spotting assay

For growth analysis of a C. glabratastrain,single colony wasinoculated in appropriate broth medium and grown for 14-16 h. Overnight grown culture was used to inoculate the test medium toan initial OD600of 0.1-0.3. Cultures were transferred to a shaker incubator set at 30°C and 200 rpm. Absorbance ofcultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at 600 nm at regular time-intervalstill 48h. Absorbancevalues were plotted with respect to time and generation time was determined from the logarithmic (log)phase of cell growth usingthefollowing formula.G = Generation time (h)T1= Initial time point taken for analysisT2= Final time point taken for analysisNf= Number of cells at time T2(1 OD600of C. glabrata corresponds to 2 X 107cells.)Ni= Number of cells at time T1(calculated from OD600value as mentioned above)

Growth analysis and determination of generation time

Bacterial strainEscherichia coli DH5αused for cloning purposewas revived on LB medium and grown at 37°C withcontinuous shaking at 200 rpm. LB medium was supplemented with appropriate antibiotics to growbacterial strains carrying plasmids. AnotherE. coli strain,BW23473,was used to rescue the Tn7transposon cassette from C. glabrataTn7insertion mutants. For plasmid DNA purification, bacterial strains were grown overnight in LB broth medium containingsuitable antibiotics

C. glabratastrains were routinely grown either in rich YPD medium or synthetically-defined YNB medium at 30°C withcontinuous shaking at 200 rpm unless otherwise stated. In general, C. glabratafrozen glycerol stocks wererevivedonYPD medium by streaking and allowed to grow for 1-2 days. C. glabratastrainsharboringthe plasmid with URA3as selectable marker were revived onCAA medium.To prepare liquid cell culture, single colony of eachC. glabratastrainwasinoculated either in YPD or YNB broth mediumand grown for 14-16 h. C. glabratastrains streaked on plates were storedat 4°C fora maximum period of2 weeks

Strains and culture conditions

Microbiological methods

To collectmacrophage-internalized yeast cellsfor RNA and protein extraction, 107THP-1 monocytes were seeded in 100 mm cell culture dishes and treated with PMA. PMA-differentiated THP-1 macrophages were infected with appropriateC. glabratastrainsto a MOIof 1:1. Equal numberof C. glabratacells wasinoculated inRPMI medium as control. Two hourspost infection,non-phagocytosed yeast cells were removed by washing THP-1 macrophages thrice with PBS. At different time points, culture dishes were washed twice with chilled PBS and 2 mlchilled sterile water was added toeach dish to lyse the macrophages. Corresponding cultures grown in RPMI medium were transferred to50 ml polypropylene tubesand transferred on ice. Lysates were collected by scrapping the macrophage monolayer and transferred to50 ml polypropylene tubes.RPMI-grown and macrophage-internalized C. glabratacells were harvested by centrifugation at 2,500g for 8 min. Macrophage cell debris were removed frommacrophage-internalized cells by repeated washing with chilled sterile water. Harvested C. glabratacells were stored at -20ºC till further use

Harvesting of macrophage-internalized C. glabratacellsfor RNA and protein extraction

undertissueculture conditionsfor 45-60min andfixed in 3.7% formaldehydeas described earlier.For DAPI staining, Vectashield mounting medium containing DAPI was used and slides were visualized under confocal microscope.For heat killing, yeast cells were harvested from 1 ml culture, washed, resuspended inPBS andwere incubated at 95°C for 5 min

PMA-treated THP-1 macrophages were infected with C. glabratacells to a MOIof 1:1 in four-chambered slides and incubated at 37°C and 5%CO2. After 1 hcoincubation, each chamber was washed thrice with PBS to eliminate extracellular yeast cellsand medium was replaced with fresh prewarmed RPMI medium containing100 nM Lysotracker Red DND-99.Infected THP-1 macrophageswere incubated

Lysotracker staining

For confocal microscopyanalysis, 5X105THP-1 cells were seeded and treatedwithPMA in 4-chambered slides. Differentiated THP-1 macrophageswere infected either with FITC-labeled or GFP-expressingC.glabratastrains to a MOIof 1:1. At different time intervals, medium was aspirated out from each chamber of 4-chambered slides and chamberes were washed twice with PBS. To fixthe infected macrophages,500 μlformaldehyde(3.7%) was added gently toeach chamber andincubated for 15 minat room temperature. Each chamber of the slide was washed twice withPBS to remove formaldehyde solution completely. To permeabilize the fixed cells, 500 μl Triton-X (0.7%) was dispensed toeach chamber and slide wasincubated at room temperature for 5 min. Chambers of the slide werewashed twice with PBS, separated from the slideusing a chamber removal device andwere air dried. Coverslips were placed onslides using Vectashield mounting mediumand bordersweresealed withnail paint. Slides werestored at 4°C until used forfluorescence imaging

Fixing of PMA-treated THP-1 macrophages

THP-1 cells were seeded ina 24-well tissue culture plate to a celldensity of 1 million cells per well,treated with PMA and were infected with yeast cells to a MOIof 10:1. Two hours post infection, cells were washed thrice with PBS and medium was replacedwith fresh prewarmed RPMI medium.Plates wereincubatedat 37ºCfor 24 h. Supernatants were collected,centrifuged at 3,000 rpm for 5 minto get rid of particulate matter,if any, andwerestored at -20°C until use. Estimation of different cytokines wasperformed using BD OptEA ELISA kits as per the supplier’s instructions

Cytokines measurement

Forinfection of THP-1 cells with single C. glabratastrain, PMA-treatedTHP-1 monocytes were seeded in 24 wellcell culture plate toa seeding density of 1 million cells per well. To prepare C. glabratacells for macrophage infection, single colony of the desiredstrain wasinoculated in YPD medium and allowed to grow for 14-16 hat 30°C. C. glabratacellsfrom 1ml overnight culture were harvested, washed with PBS andcell density was adjustedto 2X107cells/ml.50 μl of thisC. glabratacell suspension wasinfectedto macrophages to a MOIof 10:1. Two hours post infection, infected THP-1 macrophages were washed thrice with PBS to removenon-phagocytosed yeast cells and medium was replacedwith fresh prewarmed medium. Atdifferent time points post infection,infected THP-1 macrophages were washed with PBS three timesandlysed in 1 mlsterilewater. Lysates were collected by scrapping the wells with a micropipette tip, diluted in PBS and appropriatelysatedilutions were platedon YPD agar medium. Plates wereincubated at 30°C and colony forming units (CFU) were counted after 1-2 days. Final CFUs per ml were determined by

multiplying CFUs with dilution factor and fold-replication was determined by dividing the CFUs obtained at 24 h time-point by 2 h CFUs

Single infection assay

THP-1 monocytes were treated with phorbol myrsitylacetate (PMA) to differentiate them to macrophages(Tsuchiya et al., 1982). For PMA treatment, THP-1 cells grown upto 70-80% confluencewere harvested from the culture dishes at 1,000 rpm for 3 min. Harvested THP-1 cells were resuspended in 5-10 ml fresh and prewarmed complete RPMI medium. 100μlof thiscell suspensionwasappropriatelydilutedinPBS and numberof viable cells was determined by trypan blue stainingusing hemocytometer. Cell suspension was diluted with prewarmed RPMI medium to a final density of 106cells/ml. PMA was added to this THP-1 cell suspension to a final concentration of 16 nM and mixedwell.PMA-treated THP-1 cellswere seeded either in 24-well cell culture plate or culture dishes and transferred to the incubator set at 37°C and 5%CO2.After 12 hincubation, medium was replaced with fresh prewarmed medium and cells wereallowed to recover for 12 h

Treatmentof THP-1 monocytic cells with phorbol myrsityl acetate

weretransferred toa sterile 100 mm cell culture dishcontaining 11 mlfresh and prewarmed completemedium andculturedin tissue culture incubatorat37°C and 5% CO2.After 12hincubation, medium was replacedwith fresh prewarmed mediumand cells were allowed to proliferate till they acquire 80% confluence

Freezer stocks of THP-1 and Lec-2 cells were prepared either in commercial cell preservation medium (Gibco) or completemedium supplemented with 10%heat inactivated serum and 10% DMSO. For cryopreservation, 5-6 million cells were resuspended in 0.5 mlfreezing medium in 2 ml cryopreservation vials,stored in an isopropanol bath and were transferred to-70°C freezer. Aftertwo days, freezer stocks were transferred to liquid nitrogen containertill further use. To revive the cells, freezer stocks were taken outfrom liquid nitrogen container and transferred immediately to37°C water bath. After2-3 min, when freezing medium hadthawed completely,cells

Cryopreservationand revival of cell lines

To isolate primary peritoneal macrophages, 6-8 week old BALB/c mice were injected with 3% (w/v) thioglycollate broth (0.55% dextrose, 0.05% sodium thioglycollate, 0.5% sodium chloride, 0.05% agar)intraperitonealy (I.P. 50 μl/g body weight). After five days of injection, mice were euthanized by CO2inhalationand peritoneal macrophages were harvested byflushing the peritoneal cavity (lavage) with 10 mlDMEM medium(Zhang et al., 2008)

Isolation of primary (peritoneal) macrophages from BALB/c mice

THP-1 andLec-2 cell lines were obtained from ATCC (American Type Culture Collection). THP-1 and Lec-2 cells were cultured and maintained in RPMI-1640 and α-MEM media,respectively, supplemented with 10% heat inactivated fetal bovine serum, 2 mM glutamine and antibiotics (100units/ml of penicillin and 100μg/ml of streptomycin). Both cell lines were maintained at 37°C and 5% CO2in Thermo-Scientific cell culture incubator. After every 2-3 days, spent medium was replaced with fresh,pre-warmed medium. For splitting the culture, cells were harvested at 1,000 rpm for 3 min. Spent medium was discarded and cells were resuspended in 4-6 ml fresh prewarmed medium. Finally, 3-4 million cells were resuspended in 12mlmedium in 100 mm culture dishes.Cellswere cultured and maintained in tissue culture incubatorat37°C and 5% CO2

Cell lines andculture conditions